Osteosarcoma is the most common primary bone malignancy that usually presents during the adolescent and young adult years when the long bones are undergoing rapid growth. At diagnosis children with osteosarcoma frequently have pulmonary micrometastases that are not detected by chest X ray or CT. Despite aggressive chemotherapy and surgical intervention, 30-40% of patients will relapse with pulmonary metastases. The 2-year disease-free survival has not changed significantly in the past 15 years. New agents and novel approaches are needed to increase the survival rate in these children. Using a nude mouse model with human osteosarcoma lung metastases, intranasal therapy with an adenoviral vector expressing interleukin-12 resulted in a dramatic decrease in the number and size of pulmonary metastases in mice. The antitumor immunity induced by this therapy is mediated primary by natural killer cells and antiangiogenic mechanisms. IL-12 has two main anti-tumor activities that are immune-dependent and immune-independent. The nude mouse model is useful for determining the immune-independent mechanisms. Recently an orthotopic, syngenic murine osteosarcoma model was developed. After the intraosseous injection of murine tumor cells, a tumor forms in bone at the site of injection. These mice go on to spontaneously develop pulmonary metastases. This model, with an intact immune system, will allow the immune-dependent mechanisms of IL-12's antitumor activity to be determined. Four specific aims will be pursued: 1) To determine the identity and timing of cells that infiltrate tumor cells after the administration of Ad.mIL-12. 2) To determine if IL-12 activates NK cells to eradicate pulmonary osteosarcoma metastases. 3) To determine the role IL-12 (the primary inducer of interferon-?) plays in stimulating macrophage and dendritic activity. 4) To determine the role T cells play in the anti-tumor activity of gene therapy with IL-12. Successful completion of these studies will result in a better understanding of the role the immune system (when activated with IL-12 and T cells receptor agonists).